Development of Protein-Based Biotherapeutics That Penetrates Cell-Membrane and Induces Anti-Lung Cancer Effect - Improved Cell-Permeable Suppressor of Cytokine Signaling (iCP-SOCS3) Proteins, Polynucleotides Encoding the Same, and Anti-Lung Cancer Compositions Comprising the Same

ABSTRACT

In principle, protein-based biotherapeutics offers a way to control biochemical processes in living cells under non-steady state conditions and with fewer off-target effects than conventional small molecule therapeutics. However, systemic protein delivery in vivo has been proven difficult due to poor tissue penetration and rapid clearance. Protein transduction exploits the ability of some cell-penetrating peptide (CPP) sequences to enhance the uptake of proteins and other macromolecules by mammalian cells. Previously developed hydrophobic CPPs, named membrane translocating sequence (MTS), membrane translocating motif (MTM) and macromolecule transduction domain (MTD), are able to deliver biologically active proteins into a variety of cells and tissues. Various cargo proteins fused to these CPPs have been used to test the functional and/or therapeutic efficacy of protein transduction. Previously, recombinant proteins consisting of suppressor of cytokine signaling 3 (CP-SOCS3) protein fused to the fibroblast growth factor (FGF) 4-derived MTM were developed to inhibit inflammation and apoptosis. However, CP-SOCS3 fusion proteins expressed in bacteria cells were hard to be purified in soluble form. To address these critical limitations, CPP sequences called advanced MTDs (aMTDs) have been developed in this art. The development of this art has been accomplished by (i) analyzing previous developed hydrophobic CPP sequences to identify specific critical factors (CFs) that affect intracellular delivery potential and (ii) constructing artificial aMTD sequences that satisfy for each critical factor. In addition, solubilization domains (SDs) have been incorporated into the aMTD-fused SOCS3 recombinant proteins to enhance solubility with corresponding increases in protein yield and cell-/tissue-permeability. These recombinant SOCS3 proteins fused to aMTD/SD having much higher solubility/yield and cell-/tissue-permeability have been named as improved cell-permeable SOCS3 (iCP-SOCS3) proteins. Previously developed CP-SOCS3 proteins fused to MTM were only tested or used as anti-inflammatory agents to treat acute liver injury. In the present art, iCP-SOCS3 proteins have been tested for use as anti-cancer agents in the treatment of neoplasia in lung. Since SOCS3 is frequently deleted in cancer cells and loss of SOCS3 promotes resistance to apoptosis and proliferation, we reasoned that iCP-SOCS3 could be used as a protein-based intracellular replacement therapy for the treatment of lung cancer. The results demonstrated in this art support this reasoning: treatment of human non-small cell lung carcinoma cells with iCP-SOCS3 results in reduced cancer cell viability, enhanced apoptosis. Furthermore, iCP-SOCS3 inhibited migration/invasion of lung cancer cells. In the present invention with iCP-SOCS3, where SOCS3 is fused to an empirically determined combination of newly developed aMTD and customized SD, macromolecule intracellular transduction technology (MITT) enabled by the advanced MTDs may provide novel protein therapy against lung cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.Provisional Application No. 62/042,493, filed on Aug. 27, 2014, in theUnited States Patent and Trademark Office, the disclosure of which isincorporated herein in its entirety by reference.

TECHNICAL FIELD

The present invention pertains to have (i) improved cell-permeable SOCS3(iCP-SOCS3) proteins as protein-based biotherapeutics, which arewell-enhanced in their ability to transport biologically active SOCS3proteins across the plasma membrane, to increase in its solubility andmanufacturing yield, and to induce anti-non-small cell lung carcinomaeffect; (ii) polynucleotides that encode the same, and (iii) anti-lungcancer compositions that comprise the same.

BACKGROUND ART

Worldwide, lung cancer is the most common cause of cancer-related deathin men and women, and was responsible for 1.56 million deaths annually.There are two main types of primary lung cancer: non-small cell lungcancer (NSCLC), small cell lung cancer (SCLC). About 85% of lung cancersare NSCLCs which is the most common type of lung cancer. Squamous cellcarcinoma, adenocarcinoma, and large cell carcinoma are all subtypes ofnon-small cell lung cancer. Cytokines including IL-6 andinterferon-gamma (IFN-

) activate the Janus kinase (JAK)/signal transducers and activators oftranscription (STAT) signaling pathway, a vital role promoting theinflammation, carcinogenesis and metastasis in the lung. STAT3, whichfunctions as an oncogene downstream of IL-6/gp130, is hyper-activated inlung cancer cells contributes to increase cell proliferation andinhibits apoptosis.

Cytokine signaling is strictly regulated by the SOCS family proteinsinduced by different classes of agonists, including cytokines, hormonesand infectious agents. Among them, SOCS1 and SOCS3 are relativelyspecific to STAT1 and STAT3, respectively. SOCS1 inhibits JAK activationthrough its N-terminal kinase inhibitory region (KIR) by the directbinding to the activation loop of JAKs, while SOCS3 binds to januskinases (JAKs)-proximal sites on the receptor through its SH2 domain andinhibits JAK activity that blocks recruitment of STAT3. Both promoteanti-inflammatory effects due to the suppression ofinflammation-inducing cytokine signaling. Furthermore, the SOCS box,another domain in SOCS proteins, interacts with E3 ubiquitin ligasesand/or couples the SH2 domain-binding proteins to theubiquitin-proteasome pathway. Therefore, SOCSs inhibit cytokinesignaling by suppressing JAK kinase activity and degrading the activatedcytokine receptor complex.

A previous study has confirmed that SOCS3 may significantly inhibit theproliferation of lung cancer cells in vitro and indicated that SOCS3 mayact as an anti-oncogene involved in the development of tumors.Furthermore, SOCS3 may regulate the movement and migration of tumorcells. Methylation-mediated silencing of SOCS3 has been reported innon-small lung cancer (NSCLC) and other human cancers. In addition tothe effect of SOCS3 in inflammation, abnormalities of the JAK/STATpathway are also associated with cancer. It has been reported thatmethylationin of CpG islands in the functional SOCS3 promoter iscorrelated with its transcription silencing in the lung cancer celllines. Restoration of SOCS3 in lung cancer cells where SOCS3 wasmethylation-silenced resulted in the down-regulation of active STAT3,induction of apoptosis, and growth suppression of cancer cells. It meansthat SOCS3 silencing is one of the important mechanisms of constitutiveactivation of the JAK/STAT pathway in cancer pathogenesis. Therefore, itcan be suggested that intracellular SOCS3 protein replacement therapymay be useful in the treatment of lung cancer.

In the previous study, recombinant SOCS3 proteins that contain acell-penetrating peptide (CPP)-membrane-translocating motif (MTM) fromfibroblast growth factor (FGF)-4 has been reported to negatively controlJAK/STAT signaling. These recombinant SOCS3 proteins inhibited STATphosphorylation, inflammatory cytokines production and MHC-II expressionin cultured and primary macrophages. In addition, SOCS3 fused to MTMprotected mice challenged with a lethal dose of the SEB super-antigen,by suppressing apoptosis and hemorrhagic necrosis in multiple organs.However, the SOCS3 proteins fused to FGF4-derived MTM displayedextremely low solubility, poor yields and relatively low cell- andtissue-permeability. Therefore, the MTM-fused SOCS3 proteins were notsuitable for further clinical development as therapeutic agents. Toovercome these limitations, improved SOCS3 recombinant proteins(iCP-SOCS3) fused to the combination of novel hydrophobic CPPs, namelyadvanced macromolecule transduction domains (aMTDs), to greatly improvethe efficiency of membrane penetrating ability in vitro and in vivo withsolubilization domains to increase their solubility and manufacturingyield when expressed and purified from bacteria cells.

In this new art of invention, aMTD/SD-fused SOCS3 recombinant proteins(iCP-SOCS3) have much improved physicochemical characteristics(solubility & yield) and functional activity (cell-/tissue-permeability)compared to the protein fused only to FGF-4-derived MTM. In addition,the newly developed iCP-SOCS3 proteins have now been demonstrated tohave therapeutic application in treating the lung cancer, exploiting theability of SOCS3 to suppress JAK/STAT signaling. The present inventionrepresents that macromolecule intracellular transduction technology(MITT) enabled by the new hydrophobic CPPs that are aMTDs may providenovel protein therapy through SOCS3-intracellular protein replacementagainst the lung cancer. These findings suggest that restoration ofSOCS3 by replenishing the intracellular SOCS3 with iCP-SOCS3 proteincreates a new paradigm for anti-cancer therapy, and the intracellularprotein replacement therapy with the SOCS3 recombinant protein fused tothe combination of aMTD and SD pair may be useful to treat the lungcancer.

SUMMARY

An aspect of the present invention relates to improved cell-permeableSOCS3 (iCP-SOCS3) capable of mediating the transduction of biologicallyactive macromolecules into live cells.

iCP-SOCS3 fused to novel hydrophobic CPPs—namely advanced macromoleculetransduction domains (aMTDs)—greatly improve the efficiency of membranepenetrating ability in vitro and in vivo of the recombinant proteins.

iCP-SOCS3 fused to solubilization domains (SDs) greatly increase intheir solubility and manufacturing yield when they are expressed andpurified in the bacteria system.

An aspect of the present invention also relates to its therapeuticapplication for delivery of a biologically active molecule to a cellinvolving a cell-permeable SOCS3 recombinant protein, where the aMTD isattached to a biologically active cargo molecule.

Other aspects of the present invention relate to an efficient use ofaMTD sequences for drug delivery, protein therapy, intracellular proteintherapy, protein replacement therapy and peptide therapy.

An aspect of the present invention provides improved cell-permeableSOCS3 as a biotherapeutics having improved solubility/yield,cell-/tissue-permeability and anti-lung cancer effects. Therefore, thiswould allow their practically effective applications in drug deliveryand protein therapy including intracellular protein therapy and proteinreplacement therapy.

BRIEF DESCRIPTION OF DRAWINGS

The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings.

FIG. 1 shows the structure of SOCS3 recombinant proteins. A schematicdiagram of the His-tagged SOCS3 recombinant protein is illustrated andconstructed according to the present invention. The his-tag for affinitypurification (white), aMTD165 (black), SOCS3 (gray) and solubilizationdomain A and B (SDA & SDB, hatched) are shown.

FIG. 2 shows the construction of expression for SOCS3 recombinantproteins This figure shows the agarose gel electrophoresis analysisshowing plasmid DNA fragments encoding SOCS3, aMTDs fused SOCS3 and SDcloned into the pET28 (+) vector according to the present invention.

FIG. 3 shows inducible expression and purification of SOCS3 recombinantproteins. Expression of SOCS3 recombinant proteins in E. coli before (−)and after (+) induction with IPTG and purification by Ni2+ affinitychromatography (P) were monitored by SDS-PAGE, and stained withCoomassie blue.

FIG. 4 shows the improvement of solubility/yield with aMTD/SD-fusion.The solubility, yield and recovery (in percent) of soluble form fromdenatured form are indicated (left). Relative yield of recombinantproteins is normalized to the yield of HS3 protein (Right).

FIG. 5 shows aMTD-mediated cell-permeability of SOCS3 recombinantproteins. RAW264.7 cells were exposed to FITC-labeled SOCS3 recombinantproteins (10

M) for 1 hr, treated with proteinase K to remove cell-associated butnon-internalized proteins and analyzed by flow cytometry. Untreatedcells (gray) and equimolar concentration of unconjugated FITC (FITConly, green)-treated cells were served as control.

FIG. 6 shows aMTD-mediated intracellular delivery and localization ofSOCS3 recombinant proteins. Each of NIH3T3 cells was incubated for 1hour at 37° C. with 10

M FITC-labeled SOCS3 protein. Cell-permeability of SOCS3 recombinantproteins was visualized by utilizing confocal microscopy LSM700 version.

FIG. 7 shows the systemic delivery of aMTD/SD-fused SOCS3 recombinantproteins In vivo. Cryosections of saline-perfused organs were preparedfrom mice 1 hr after intraperitoneal injection of FITC only or 600

g FITC-conjugated recombinant SOCS3 proteins, and were analyzed byfluorescence microscopy.

FIG. 8 shows the structure of SDB-fused SOCS3 recombinant protein. Aschematic diagram of the SOCS3 recombinant protein is illustrated andconstructed according to the present invention. The his-tag for affinitypurification (white), SOCS3 (gray) and solubilization domain B (SDB,hatched) are shown.

FIG. 9 shows the expression, purification and determination ofsolubility/yield of SD-fused SOCS3 recombinant protein. Expression ofSOCS3 recombinant proteins in E. coli before (−) and after (+) inductionwith IPTG and purification by Ni2+ affinity chromatography (P) weremonitored by SDS-PAGE, and stained with Coomassie blue (Left, top). Thesolubility, yield and recovery (in percent) of soluble form fromdenatured form are indicated (Left, bottom). Relative yield ofrecombinant proteins is normalized to the yield of HS3 protein (Right).

FIG. 10 shows the mechanism of aMTD-mediated SOCS3 protein uptake intocells. (A-D) RAW264.7 cells were treated with 100 mM EDTA for 3 hrs (A),5 mg/ml Proteinase K for 10 mins (B), 20 mM taxol for 30 mins (C), or 10μM antimycin for 2 hrs either without or with 1 mM supplemental ATP for3 hrs. Cells were exposed for 1 hr to 10

M FITC-labeled HS3 (black), -HS3B (blue) or -HM165S3B (red), treatedwith proteinase K for 20 mins, and analyzed by flow cytometry. Untreatedcells (gray) and equimolar concentration of unconjugated FITC (FITConly, green)-treated cells were served as control. (E) RAW264.7 cellswere exposed for the indicated times to 10 μM FITC-labeled HS3 (black),-HS3B (blue) or -HM165S3B (red), treated with proteinase K, and analyzedby flow cytometry.

FIG. 11 shows aMTD-mediated cell-to-cell delivery. RAW264.7 cellsexposed to 10 μM FITC-HS3B or FITC-HM165S3B for 2 hrs, were mixed withnon-treated RAW264.7 cells pre-stained with Cy5.5 labeled anti-CD14antibody, and analyzed by flow cytometry (left, top). The top (right)panel shows a mixture of double negative cells (cells exposed toFITC-HS3B that did not incorporate the protein) and single positiveCy5.5 labeled cells; whereas, second panel from the left containsFITC-Cy5.5 double-positive cells generated by the transfer ofFITC-HM165S3B to Cy5.5 labeled cells and the remaining FITC and Cy5.5single-positive cells. The bottom panels show FITC fluorescence profilesof cell populations before mixing (coded as before) and 1 hr after thesame cells were mixed with Cy5.5-labeled cells.

FIG. 12 shows the inhibition of STAT phosphorylation induced by IFN-

. Inhibition of STAT1 phosphorylation detected by immunoblottinganalysis. The levels of phosphorylated STAT1 and STAT3 untreated andtreated with IFN-

were compared to the levels in IFN-

-treated RAW 264.7 cells that were pulsed with 10

M of indicated proteins.

FIG. 13 shows the inhibition of cytokines secretion induced by LPS.Inhibition of TNF-

and IL-6 expression by recombinant SOCS3 proteins in primary macrophagesisolated from peritoneal exudates of C3H/HeJ mice. Error barsindicate+s.d. of the mean value derived from each assay done intriplicate.

FIG. 14 shows the cell-permeability of iCP-SOCS3 (HM165S3B) in lungcancer cells. A549 lung cancer cells were exposed to FITC-labeled SOCS3recombinant proteins (10

M) for 1 hr, treated with proteinase K to remove cell-associatedproteins for 20 mins, and analyzed by flow cytometry. Untreated cells(gray) and equimolar concentration of unconjugated FITC (FITC only,green)-treated cells were served as control.

FIG. 15 shows the tissue distribution of iCP-SOCS3 (HM165S3B) into lungtissue. Cryosections of saline-perfused organs were prepared from mice 1hr after intraperitoneal injection of FITC only or 600

g FITC-conjugated recombinant SOCS3 proteins, and were analyzed byfluorescence microscopy.

FIG. 16 shows the inhibition of proliferation in lung cancer cells withiCP-SOCS3. A549 lung cancer cells were seeded in 96 well plates. Nextday, cells were treated with DMEM (V), HS3 (1), HM165S3 (2), HM165S3A(3) or HM165S3B (4) recombinant proteins for 96 hrs in the presence ofserum (2%). Cell viability was evaluated with the CellTiter-Glo CellViability Assay.

FIG. 17 shows the induction of apoptosis in lung cancer cells withiCP-SOCS3. A549 lung cancer cells were treated for 24 hrs with 10 μMHS3B or HM165S3B proteins and apoptotic cells were visualized by TUNELstaining.

FIG. 18 shows the stimulation of apoptosis in lung cancer cells withiCP-SOCS3. A549 lung cancer cells were treated for 24 hr with 10 μM HS3Bor HM165S3B proteins and analyzed by flow cytometry of cells stainedwith annexin-V and 7-AAD.

FIG. 19 shows the inhibition of migration in lung cancer cells withiCP-SOCS3. A549 lung cancer cells were grown to 100% confluence andthese procedures were performed on wound-healing assays. The wound areaswere examined and photographed at 0 and 48 hrs post-wounding.

FIG. 20 shows the inhibition of migration/invasion in lung cancer cellswith iCP-SOCS3. A549 lung cancer cells were treated with SOCS3recombinant proteins for 24 hrs, and migration/invasion were measured byTranswell assay. The data shown are representative of three independentexperiments. **, p<0.01.

DETAILED DESCRIPTION

In this invention, it has been hypothesized that exogenouslyadministered SOCS3 proteins could compensate for the apparent inabilityof endogenously expressed members of this physiologic regulator tointerrupt constitutively active cancer-initiating JAK/STAT signaling andexcessive cell cycle, resulting in the inhibition of the tumorigenesis.To prove our hypothesis, the SOCS3 recombinant proteins were fused tonovel hydrophobic CPPs called aMTDs to improve theircell-/tissue-permeability, additionally adopted solubilization domainsto increase their solubility/yield in physiological condition, and thentested whether exogenous administration of SOCS3 proteins canreconstitute their endogenous stores and restore their basic function asthe negative feedback regulator that attenuates JAK/STAT signaling. Thisart of invention has demonstrated “intracellular protein therapy” bydesigning and introducing cell-permeable form of SOCS3 that has a greatpotential of anti-cancer therapeutic applicability in lung cancer.

1. Novel Hydrophobic Cell-Penetrating Peptides—Advanced MacromoleculeTransduction Domains

To address the limitation of previously developed hydrophobic CPPs,novel sequences have been developed. To design new hydrophobic CPPs forintracellular delivery of cargo proteins such as SOCS3, identificationof optimal common sequence and/or homologous structural determinants,namely critical factors (CFs), had been crucial. To do it, thephysicochemical characteristics of previously published hydrophobic CPPswere analyzed. To keep the similar mechanism on cellular uptake, allCPPs analyzed were hydrophobic region of signal peptide (HRSP)-derivedCPPs (e.g. MTS and MTD).

(1) Basic Characteristics of CPPs Sequence.

These 17 hydrophobic CPPs published from 1995 to 2014 have been analyzedfor their 11 different characteristics—sequence, amino acid length,molecular weight, pl value, bending potential, rigidity/flexibility,structural feature, hydropathy, residue structure, amino acidcomposition, and secondary structure of the sequences. Twopeptide/protein analysis programs were used (ExPasy:http://web.expasy.org/protparam/, SoSui:http://harrier.nagahama-i-bio.ac.jp/sosui/sosui_submit.html) todetermine various indexes, structural features of the peptide sequencesand to design new sequence. Followings are important factors analyzed.

Average length, molecular weight and pl value of the peptides analyzedwere 10.8±2.4, 1,011±189.6 and 5.6±0.1, respectively.

(2) Bending Potential (Proline Position: PP)

Bending potential (Bending or No-Bending) was determined based on thefact whether proline (P) exists and/or where the amino acid(s) providingbending potential to the peptide in recombinant protein is/are located.Proline differs from the other common amino acids in that its side chainis bonded to the backbone nitrogen atom as well as the alpha-carbonatom. The resulting cyclic structure markedly influences proteinarchitecture which is often found in the bends of folded peptide/proteinchain. Eleven out of 17 were determined as ‘Bending’ peptide which meansthat proline should be present in the middle of sequence for peptidebending and/or located at the end of the peptide for protein bending. Asindicated above, peptide sequences could penetrate the plasma membranein a “bent” configuration. Therefore, bending or no-bending potential isconsidered as one of the critical factors for the improvement of currenthydrophobic CPPs.

(3) Rigidity/Flexibility (Instability Index: II)

Since one of the crucial structural features of any peptide is based onthe fact whether the motif is rigid or flexible, which is an intactphysicochemical characteristic of the peptide sequence, instabilityindex (II) of the sequence was determined. The index value representingrigidity/flexibility of the peptide was extremely varied (8.9-79.1), butaverage value was 40.1±21.9 which suggested that the peptide should besomehow flexible, but not too rigid or flexible.

(4) Hydropathy (Grand Average of Hydropathy: GRAVY) and StructuralFeature (Aliphatic Index: AI)

Alanine (V), valine (V), leucine (L) and isoleucine (I) containaliphatic side chain and are hydrophobic—that is, they have an aversionto water and like to cluster. These amino acids having hydrophobicityand aliphatic residue enable them to pack together to form compactstructure with few holes. Analyzed peptide sequence showed that allcomposing amino acids were hydrophobic (A, V, L and I) except glycine(G) in only one out of 17 and aliphatic (A, V, L, I, and P). Theirhydropathic index (Grand Average of Hydropathy: GRAVY) and aliphaticindex (AI) were 2.5±0.4 and 217.9±43.6, respectively.

(5) Secondary Structure (α-Helix)

As explained above, the CPP sequences may be supposed to penetrate theplasma membrane directly after inserting into the membranes in a “bent”configuration with hydrophobic sequences adopting an α-helicalconformation. In addition, our analysis strongly indicated that bendingpotential was crucial. Therefore, structural analysis of the peptidesconducted to determine whether the sequence was to form helix or not.Nine peptides were helix and 8 were not. It seems to suggest that helixstructure may not be required.

(6) Determination of Critical Factors (CFs)

In the 11 characteristics analyzed, the following 6 are selected namely“Critical Factors (CFs)” for the development of new hydrophobicCPPs—advanced MTDs: i) amino acid length, ii) bending potential (prolinepresence and location), iii) rigidity/flexibility (instability index:II), iv) structural feature (aliphatic index: AI), v) hydropathy (GRAVY)and vi) amino acid composition/residue structure (hydrophobic andaliphatic A/a).

1-2. Analysis of Selected Hydrophobic CPPs to Optimize ‘CriticalFactors’

Since the analyzed data of the 17 different hydrophobic CPPs (analysisA) previously developed during the past 2 decades showed high variationand were hard to make common- or consensus-features, additional analysisB and C was also conducted to optimize the critical factors for betterdesign of improved CPPs-aMTDs.

In analysis B, 8 CPPs used with each cargo in vivo were selected. Lengthwas 11±3.2, but 3 out of 8 CPPs possessed little bending potential.Rigidity/Flexibility was 41±15, but removing one [MTD85: rigid, withminimal (II: 9.1)] of the peptides increased the overall instabilityindex to 45.6±9.3. This suggested that higher flexibility (40 or higherII) is potentially be better. All other characteristics of the 8 CPPswere similar to the analysis A, including structural feature andhydropathy.

To optimize the ‘Common Range and/or Consensus Feature of CriticalFactor’ for the practical design of aMTDs and the random peptides, whichwere to prove that the ‘Critical Factors’ determined in the analysis A,B and C were correct to improve the current problems of hydrophobicCPPs—protein aggregation, low solubility/yield, and poorcell/tissue-permeability of the recombinant proteins fused to theMTS/MTM or MTD, and non-common sequence and non-homologous structure ofthe peptides, empirically selected peptides were analyzed for theirstructural features and physicochemical factor indexes.

The peptides which did not have a bending potential, rigid or tooflexible sequences (too low or too high Instability Index), or too lowor too high hydrophobic CPP were unselected, but secondary structure wasnot considered because helix structure of sequence was not required. 8selected CPP sequences that could provide a bending potential and higherflexibility were finally analyzed. Common amino acid length is 12(11.6±3.0). Proline should be presence in the middle of and/or the endof sequence. Rigidity/Flexibility (II) is 45.5-57.3 (Avg: 50.1±3.6). AIand GRAVY representing structural feature and hydrophobicity of thepeptide are 204.7±37.5 and 2.4±0.3, respectively. All peptides areconsisted with hydrophobic and aliphatic amino acids (A, V, L, I, andP). Therefore, analysis C was chosen as a standard for the new design ofnew hydrophobic CPPs (TABLE 1).

-   -   1. Amino Acid Length: 9-13    -   2. Bending Potential (Proline Position: PP): Proline presences        in the middle (from 5′ to 8′ amino acid) and at the end of        sequence    -   3. Rigidity/Flexibility (Instability Index: II): 40-60    -   4. Structural Feature (Aliphatic Index: AI): 180-220    -   5. Hydropathy (Grand Average of Hydropathy: GRAVY): 2.1-2.6    -   6. Amino Acid Composition: Hydrophobic and Aliphatic amino        acids—A, V, L, I and P

TABLE 1 [Universal Structure of Newly Develop Hydrophobic CPPs]Summarized Critical Factors of aMTD Newly Designed CPPs Critical FactorRange Bending Potential Proline presences in the middle (5′, 6′, 7′ or8′) (Proline Position: PP) and at the end (12′) of peptidesRigidity/Flexibility 40-60 (Instability Index: II) Structural Feature180-220 (Aliphatic Index: Al) Hydropathy 2.1-2.6 (Grand Average ofHydropathy GRAVY) Length  9-13 (Number of Amino Acid) Amino acidComposition A, V, I, L, P1-3. Determination of Critical Factors for Development of aMTDs

For confirming the validity of 6 critical factors providing theoptimized cell-/tissue-permeability. All 240 aMTD sequences have beendesigned and developed based on six critical factors (TABLES 2-1 to2-6). (The aMTD amino sequences are SEQ ID NOS: 1 to 240, and the aMTDnucleotide sequences are SEQ ID NOS: 241 to 480.)

All 240 aMTDs (hydrophobic, flexible, bending, aliphatic and helical 12a/a-length peptides) were practically confirmed by their quantitativeand visual cell-permeability. To determine the cell-permeability ofaMTDs and random peptides which do not satisfy one or more criticalfactors have also been designed and tested. Relative cell-permeabilityof 240 aMTDs to the negative control (random peptide, hydrophilic &non-aliphatic 12A/a length peptide) was significantly increased by up to164 fold, with average increase of 19.6±1.6. Moreover, compared withreference CPPs (MTM and MTD), novel 240 aMTDs averaged of 13±1.1(maximum 109.9) and 6.6±0.5 (maximum 55.5) fold highercell-permeability, respectively. As a result, there were vividassociation of cell-permeability of the peptides and critical factors.According to the result from the newly designed and tested novel 240aMTDs, the empirically optimized critical factors are provided below.

-   -   1. Amino Acid Length: 12    -   2. Bending Potential (Proline Position: PP)    -   : Proline presences in the middle (from 5′ to 8′ amino acid) and        at the end of sequence    -   3. Rigidity/Flexibility (Instability Index: II): 41.3-57.3    -   4. Structural Feature (Aliphatic Index: AI): 187.5-220.0    -   5. Hydropathy (Grand Average of Hydropathy: GRAVY): 2.2-2.6    -   6. Amino Acid Composition: Hydrophobic and Aliphatic amino        acids—A, V, L, I and P

TABLE 2-1 [Newly Developed Hydrophobic CPPs-240 aMTDs That All Critical Factors AreConsidered and Satisfied (Sequence ID No. 1-46)] Sequence Rigidity/Sturctural ID Flexibility Feature Hydropathy Residue Number aMTDSequences Length (II) (Al) (GRAVY) Structure 1 1 AAALAPVVLALP 12 57.3187.5 2.1 Aliphatic 2 2 AAAVPLLAVVVP 12 41.3 195.0 2.4 Aliphatic 3 3AALLVPAAVLAP 12 57.3 187.5 2.1 Aliphatic 4 4 ALALLPVAALAP 12 57.3 195.82.1 Aliphatic 5 5 AAALLPVALVAP 12 57.3 187.5 2.1 Aliphatic 6 11VVALAPALAALP 12 57.3 187.5 2.1 Aliphatic 7 12 LLAAVPAVLLAP 12 57.3 211.72.3 Aliphatic 8 13 AAALVPVVALLP 12 57.3 203.3 2.3 Aliphatic 9 21AVALLPALLAVP 12 57.3 211.7 2.3 Aliphatic 10 22 AVVLVPVLAAAP 12 57.3195.0 2.4 Aliphatic 11 23 VVLVLPAAAAVP 12 57.3 195.0 2.4 Aliphatic 12 24IALAAPALIVAP 12 50.2 195.8 2.2 Aliphatic 13 25 IVAVAPALVALP 12 50.2203.3 2.4 Aliphatic 14 42 VAALPVVAVVAP 12 57.3 186.7 2.4 Aliphatic 15 43LLAAPLVVAAVP 12 41.3 187.5 2.1 Aliphatic 16 44 ALAVPVALLVAP 12 57.3203.3 2.3 Aliphatic 17 61 VAALPVLLAALP 12 57.3 211.7 2.3 Aliphatic 18 62VALLAPVALAVP 12 57.3 203.3 2.3 Aliphatic 19 63 AALLVPALVAVP 12 57.3203.3 2.3 Aliphatic 20 64 AIVALPVAVLAP 12 50.2 203.3 2.4 Aliphatic 21 65IAIVAPVVALAP 12 50.2 203.3 2.4 Aliphatic 22 81 AALLPALAALLP 12 57.3204.2 2.1 Aliphatic 23 82 AVVLAPVAAVLP 12 57.3 195.0 2.4 Aliphatic 24 83LAVAAPLALALP 12 41.3 195.8 2.1 Aliphatic 25 84 AAVAAPLLLALP 12 41.3195.8 2.1 Aliphatic 26 85 LLVLPAAALAAP 12 57.3 195.8 2.1 Aliphatic 27101 LVALAPVAAVLP 12 57.3 203.3 2.3 Aliphatic 28 102 LALAPAALALLP 12 57.3204.2 2.1 Aliphatic 29 103 ALIAAPILALAP 12 57.3 204.2 2.2 Aliphatic 30104 AVVAAPLVLALP 12 41.3 203.3 2.3 Aliphatic 31 105 LLALAPAALLAP 12 57.3204.1 2.1 Aliphatic 32 121 AIVALPALALAP 12 50.2 195.8 2.2 Aliphatio 33123 AAIIVPAALLAP 12 50.2 195.8 2.2 Aliphatic 34 124 IAVALPALIAAP 12 50.3195.8 2.2 Aliphatic 35 141 AVIVLPALAVAP 12 50.2 203.3 2.4 Aliphatio 36143 AVLAVPAVLVAP 12 57.3 195.0 2.4 Aliphatic 37 144 VLAIVPAVALAP 12 50.2203.3 2.4 Aliphatic 38 145 LLAVVPAVALAP 12 57.3 203.3 2.3 Aliphatic 39161 AVIALPALIAAP 12 57.3 195.8 2.2 Aliphatic 40 162 AVVALPAALIVP 12 50.2203.3 2.4 Aliphatic 41 163 LALVLPAALAAP 12 57.3 195.8 2.1 Aliphatic 42164 LAAVLPALLAAP 12 57.3 195.8 2.1 Aliphatic 43 165 ALAVPVALAIVP 12 50.2203.3 2.4 Aliphatic 44 182 ALIAPVVALVAP 12 57.3 203.3 2.4 Aliphatic 45183 LLAAPVVIALAP 12 57.3 211.6 2.4 Aliphatic 46 184 LAAIVPAIIAVP 12 50.2211.6 2.4 Aliphatic

TABLE 2-2 [Newly Developed Hydrophobic CPPs-240 aMTDs That All Critical FactorsAre Considered and Satisfied (Sequence ID No. 47-92)] Sequence Rigidity/Sturctural ID Flexibility Feature Hydropathy Residue Number aMTDSequences Length (II) (Al) (GRAVY) Structure 47 185 AALVLPLIIAAP 12 41.3220.0 2.4 Aliphatic 48 201 LALAVPALAALP 12 57.3 195.5 2.1 Aliphatic 49204 LIAALPAVAALP 12 57.3 195.5 2.2 Aliphatic 50 205 ALALVPAIAALP 12 57.3195.8 2.2 Aliphatic 51 221 AAILAPIVALAP 12 50.2 195.8 2.2 Aliphatic 52222 ALLIAPAAVIAP 12 57.3 195.8 2.2 Aliphatic 53 223 AILAVPIAVVAP 12 57.3203.3 2.4 Aliphatic 54 224 ILAAVPIALAAP 12 57.3 195.8 2.2 Aliphatic 55225 VAALLPAAAVLP 12 57.3 187.5 2.1 Aliphatic 56 241 AAAVVPVLLVAP 12 57.3195.0 2.4 Aliphatic 57 242 AALLVPALVAAP 12 57.3 187.5 2.1 Aliphatic 58243 AAVLLPVALAAP 12 57.3 187.5 2.1 Aliphatic 59 245 AAALAPVLALVP 12 57.3187.5 2.1 Aliphatic 60 261 LVLVPLLAAAAP 12 41.3 211.6 2.3 Aliphatic 61262 ALIAVPAIIVAP 12 50.2 211.6 2.4 Aliphatic 62 263 ALAVIPAAAILP 12 54.9195.8 2.2 Aliphatic 63 264 LAAAPVVIVIAP 12 50.2 203.3 2.4 Aliphatic 64265 VLAIAPLLAAVP 12 41.3 211.6 2.3 Aliphatic 65 281 ALIVLPAAVAVP 12 50.2203.3 2.4 Aliphatic 66 282 VLAVAPALIVAP 12 50.2 203.3 2.4 Aliphatic 67283 AALLAPALIVAP 12 50.2 195.8 2.2 Aliphatic 68 284 ALIAPAVALIVP 12 50.2211.7 2.4 Aliphatic 69 285 AIVLLPAAVVAP 12 50.2 203.3 2.4 Aliphatic 70301 VIAAPVLAVLAP 12 57.3 203.3 2.4 Aliphatic 71 302 LALAPALALLAP 12 57.3204.2 2.1 Aliphatic 72 304 AIILAPIAAIAP 12 57.3 204.2 2.3 Aliphatic 73305 IALAAPILLAAP 12 57.3 204.2 2.2 Aliphatic 74 321 IVAVALPALAVP 12 50.2203.3 2.3 Aliphatic 75 322 VVAIVLPALAAP 12 50.2 203.3 2.3 Aliphatic 76323 IVAVALPVALAP 12 50.2 203.3 2.3 Aliphatic 77 324 IVAVALPAALVP 12 50.2203.3 2.3 Aliphatic 78 325 IVAVALPAVALP 12 50.2 203.3 2.3 Aliphatic 79341 IVAVALPAVLAP 12 50.2 203.3 2.3 Aliphatic 80 342 VIVALAPAVLAP 12 50.2203.3 2.3 Aliphatic 81 343 IVAVALPALVAP 12 50.2 203.3 2.3 Aliphatic 82345 ALLIVAPVAVAP 12 50.2 203.3 2.3 Aliphatic 83 361 AVVIVAPAVIAP 12 50.2195.0 2.4 Aliphatic 84 363 AVLAVAPALIVP 12 50.2 203.3 2.3 Aliphatic 85364 LVAAVAPALIVP 12 50.2 203.3 2.3 Aliphatic 86 365 AVIVVAPALLAP 12 50.2203.3 2.3 Aliphatic 87 381 VVAIVLPAVAAP 12 50.2 195.0 2.4 Aliphatic 88382 AAALVIPAILAP 12 54.9 195.8 2.2 Aliphatic 89 383 VIVALAPALLAP 12 50.2211.6 2.3 Aliphatic 90 384 VIVAIAPALLAP 12 50.2 211.6 2.4 Aliphatic 91385 IVAIAVPALVAP 12 50.2 203.3 2.4 Aliphatic 92 401 AALAVIPAAILP 12 54.9195.8 2.2 Aliphatic

TABLE 2-3 [Newly Developed Hydrophobic CPPs-240 aMTDs That All Critical Factors AreConsidered and Satisfied (Sequence ID No. 93-138)] Sequence Rigidity/Sturctural ID Flexibility Feature Hydropathy Residue Number aMTDSequences Length (II) (Al) (GRAVY) Structure 93 402 ALAAVIPAAILP 12 54.9195.8 2.2 Aliphatic 94 403 AAALVIPAAILP 2 54.9 195.6 2.2 Aliphatic 95404 LAAAVIPAAILP 2 54.9 195.8 2.2 Aliphatic 96 405 LAAAVIPVAILP 12 54.9211.7 2.4 Aliphatic 97 421 AAILAAPLIAVP 12 57.3 195.8 2.2 Aliphatic 98422 VVAILAPLLAAP 12 57.3 211.7 2.4 Aliphatic 99 424 AVVVAAPVLALP 12 57.3195.0 2.4 Aliphatic 100 425 AVVAIAPVLALP 12 57.3 203.3 2.4 Aliphatic 101442 ALAALVPAVLVP 12 57.3 203.3 2.3 Aliphatic 102 443 ALAALVPVALVP 1257.3 203.3 2.3 Aliphatic 103 444 LAAALVPVALVP 12 57.3 203.3 2.3Aliphatic 104 445 ALAALVPALVVP 12 57.3 203.3 2.3 Aliphatic 105 461IAAVIVPAVALP 12 50.2 203.3 2.4 Aliphatic 106 462 IAAVLVPAVALP 12 57.3203.3 2.4 Aliphatic 107 463 AVAILVPLLAAP 12 57.3 211.7 2.4 Aliphatic 108464 AVVILVPLAAAP 12 57.3 203.3 2.4 Aliphatic 109 465 IAAVIVPVAALP 1250.2 203.3 2.4 Aliphatic 110 481 AIAIAIVPVALP 12 50.2 211.6 2.4Aliphatic 111 482 ILAVAAIPVAVP 12 54.9 203.3 2.4 Aliphatic 112 483ILAAAIIPAALP 12 54.9 204.1 2.2 Aliphatic 113 484 LAVVLAAPAIVP 12 50.2203.3 2.4 Aliphatic 114 485 AILAAIVPLAVP 12 50.2 211.6 2.4 Aliphatic 115501 VIVALAVPALAP 12 50.2 203.3 2.4 Aliphatic 116 502 AIVALAVPVLAP 1250.2 203.3 2.4 Aliphatic 117 503 AAIIIVLPAALP 12 50.2 220.0 2.4Aliphatic 118 504 LIVALAVPALAP 12 50.2 211.7 2.4 Aliphatic 119 505AIIIVIAPAAAP 12 50.2 195.8 2.3 Aliphatic 120 521 LAALIVVPAVAP 12 50.2203.3 2.4 Aliphatic 121 522 ALLVIAVPAVAP 12 57.3 203.3 2.4 Aliphatic 122524 AVALIVVPALAP 12 50.2 203.3 2.4 Aliphatic 123 525 ALAIVVAPVAVP 1250.2 195.0 2.4 Aliphatic 124 541 LLALIIAPAAAP 12 57.3 204.1 2.1Aliphatic 125 542 ALALIIVPAVAP 12 50.2 211.6 2.4 Aliphatic 126 543LLAALIAPAALP 12 57.3 204.1 2.1 Aliphatic 127 544 IVALIVAPAAVP 12 43.1203.3 2.4 Aliphatic 128 545 VVLVLAAPAAVP 12 57.3 195.0 2.3 Aliphatic 129561 AAVAIVLPAVVP 12 50.2 195.0 2.4 Aliphatic 130 562 ALIAAIVPALVP 1250.2 211.7 2.4 Aliphatic 131 563 ALAVIVVPALAP 12 50.2 203.3 2.4Aliphatic 132 564 VAIALIVPALAP 12 50.2 211.7 2.4 Aliphatic 133 565VAIVLVAPAVAP 12 50.2 195.0 2.4 Aliphatic 134 582 VAVALIVPALAP 12 50.2203.3 2.4 Aliphatic 135 583 AVILALAPIVAP 12 50.2 211.6 2.4 Aliphatic 136585 ALIVAIAPALVP 12 50.2 211.6 2.4 Aliphatic 137 601 AAILIAVPIAAP 1257.3 195.8 2.3 Aliphatic 138 602 VIVALAAPVLAP 12 50.2 203.3 2.4Aliphatic

TABLE 2-4 [Newly Developed Hydrophobic CPPs-240 aMTDs That All Critical Factors AreConsidered and Satisfied (Sequence ID No. 139-184)] Sequence Rigidity/Sturctural ID Flexibility Feature Hydropathy Residue Number aMTDSequences Length (II) (Al) (GRAVY) Structure 139 603 VLVALAAPVIAP 1257.3 203.3 2.4 Aliphatic 140 604 VALIAVAPAVVP 12 57.3 195.0 2.4Aliphatic 141 605 VIAAVLAPVAVP 12 57.3 195.0 2.4 Aliphatic 142 622ALIVLAAPVAVP 12 50.2 203.3 2.4 Aliphatic 143 623 VAAAIALPAIVP 12 50.2187.5 2.3 Aliphatic 144 625 ILAAAAAPLIVP 12 50.2 195.8 2.2 Aliphatic 145643 LALVLAAPAIVP 12 50.2 211.6 2.4 Aliphatic 146 645 ALAVVALPAIVP 1250.2 203.3 2.4 Aliphatic 147 661 AAILAPIVAALP 12 50.2 195.8 2.2Aliphatic 148 664 ILIAIAIPAAAP 12 54.9 204.1 2.3 Aliphatic 149 665LAIVLAAPVAVP 12 50.2 203.3 2.3 Aliphatic 150 666 AAIAIIAPAIVP 12 50.2195.8 2.3 Aliphatic 151 667 LAVAIVAPALVP 12 50.2 203.3 2.3 Aliphatic 152683 LAIVLAAPAVLP 12 50.2 211.7 2.4 Aliphatic 153 684 AAIVLALPAVLP 1250.2 211.7 2.4 Aliphatic 154 685 ALLVAVLPAALP 12 57.3 211.7 2.3Aliphatic 155 686 AALVAVLPVALP 12 57.3 203.3 2.3 Aliphatic 156 687AILAVALPLLAP 12 57.3 220.0 2.3 Aliphatic 157 703 IVAVALVPALAP 12 50.2203.3 2.4 Aliphatic 158 705 IVAVALLPALAP 12 50.2 211.7 2.4 Aliphatic 159706 IVAVALLPAVAP 12 50.2 203.3 2.4 Aliphatic 160 707 IVALAVLPAVAP 1250.2 203.3 2.4 Aliphatic 161 724 VAVLAVLPALAP 12 57.3 203.3 2.3Aliphatic 162 725 IAVLAVALAVLP 12 57.3 203.3 2.3 Aliphatic 163 726LAVAIIAPAVAP 12 57.3 187.5 2.2 Aliphatic 164 727 VALAIALPAVLP 12 57.3211.6 2.3 Aliphatic 165 743 AIAIALVPVALP 12 57.3 211.6 2.4 Aliphatic 166744 AAVVIVAPVALP 12 50.2 195.0 2.4 Aliphatic 167 746 VAIIVVAPALAP 1250.2 203.3 2.4 Aliphatic 168 747 VALLAIAPALAP 12 57.3 195.8 2.2Aliphatic 169 763 VAVLIAVPALAP 12 57.3 203.3 2.3 Aliphatic 170 764AVALAVLPAVVP 12 57.3 195.0 2.3 Aliphatic 171 765 AVALAVVPAVLP 12 57.3195.0 2.3 Aliphatic 172 766 IVVIAVAPAVAP 12 50.2 195.0 2.4 Aliphatic 173767 IVVAAVVPALAP 12 50.2 195.0 2.4 Aliphatic 174 783 IVALVPAVAIAP 1250.2 203.3 2.5 Aliphatic 175 784 VAALPAVALVVP 12 57.3 195.0 2.4Aliphatic 176 786 LVAIAPLAVLAP 12 41.3 211.7 2.4 Aliphatic 177 787AVALVPVIVAAP 12 50.2 195.0 2.4 Aliphatic 178 788 AIAVAIAPVALP 12 57.3187.5 2.3 Aliphatic 179 803 AIALAVPVLALP 12 57.3 211.7 2.4 Aliphatic 180805 LVLIAAAPIALP 12 41.3 220.0 2.4 Aliphatic 181 806 LVALAVPAAVLP 1257.3 203.3 2.3 Aliphatic 182 807 AVALAVPALVLP 12 57.3 203.3 2.3Aliphatic 183 808 LVVLAAAPLAVP 12 41.3 203.3 2.3 Aliphatic 184 809LIVLAAPALAAP 12 50.2 195.8 2.2 Aliphatic

TABLE 2-5 [Newly Developed Hydrophobic CPPs-240 aMTDs That All Critical Factors AreConsidered and Satisfied (Sequence ID No. 185-230)] Sequence Rigidity/Sturctural ID Flexibility Feature Hydropathy Residue Number aMTDSequences Length (II) (Al) (GRAVY) Structure 185 810 VIVLAAPALAAP 1250.2 187.5 2.2 Aliphatic 186 811 AVVLAVPALAVP 12 57.3 195.0 2.3Aliphatic 187 824 LIIVAAAPAVAP 12 50.2 187.5 2.3 Aliphatic 188 825IVAVIVAPAVAP 12 43.2 195.0 2.5 Aliphatic 189 826 LVALAAPIIAVP 12 41.3211.7 2.4 Aliphatic 190 827 IAAVLAAPALVP 12 57.3 187.5 2.2 Aliphatic 191828 IALLAAPIIAVP 12 41.3 220.0 2.4 Aliphatic 192 829 AALALVAPVIVP 1250.2 203.3 2.4 Aliphatic 193 830 IALVAAPVALVP 12 57.3 203.3 2.4Aliphatic 194 831 IIVAVAPAAIVP 12 43.2 203.3 2.5 Aliphatic 195 832AVAAIVPVIVAP 12 43.2 195.0 2.5 Aliphatic 196 843 AVLVLVAPAAAP 12 41.3219.2 2.5 Aliphatic 197 844 VVALLAPLIAAP 12 41.3 211.8 2.4 Aliphatic 198845 AAVVIAPLLAVP 12 41.3 203.3 2.4 Aliphatic 199 846 IAVAVAAPLLVP 1241.3 203.3 2.4 Aliphatic 200 847 LVAIVVLPAVAP 12 50.2 219.2 2.6Aliphatic 201 848 AVAIVVLPAVAP 12 50.2 195.0 2.4 Aliphatic 202 849AVILLAPLIAAP 12 57.3 220.0 2.4 Aliphatic 203 850 LVIALAAPVALP 12 57.3211.7 2.4 Aliphatic 204 851 VLAVVLPAVALP 12 57.3 219.2 2.5 Aliphatic 205852 VLAVAAPAVLLP 12 57.3 203.3 2.3 Aliphatic 206 863 AAVVLLPIIAAP 1241.3 211.7 2.4 Aliphatic 207 864 ALLVIAPAIAVP 12 57.3 211.7 2.4Aliphatic 208 865 AVLVIAVPAIAP 12 57.3 203.3 2.5 Aliphatic 209 867ALLVVIAPLAAP 12 41.3 211.7 2.4 Aliphatic 210 868 VLVAAILPAAIP 12 54.9211.7 2.4 Aliphatic 211 870 VLVAAVLPIAAP 12 41.3 203.3 2.4 Aliphatic 212872 VLAAAVLPLVVP 12 41.3 219.2 2.5 Aliphatic 213 875 AIAIVVPAVAVP 1250.2 195.0 2.4 Aliphatic 214 877 VAIIAVPAVVAP 12 57.3 195.0 2.4Aliphatic 215 878 IVALVAPAAVVP 12 50.2 195.0 2.4 Aliphatic 216 879AAIVLLPAVVVP 12 50.2 219.1 2.5 Aliphatic 217 881 AALIVVPAVAVP 12 50.2195.0 2.4 Aliphatic 218 882 AIALVVPAVAVP 12 57.3 195.0 2.4 Aliphatic 219883 LAIVPAAIAALP 12 50.2 195.8 2.2 Aliphatic 220 885 LVAIAPAVAVLP 1257.3 203.3 2.4 Aliphatic 221 887 VLAVAPAVAVLP 12 57.3 195.0 2.4Aliphatic 222 888 ILAVVAIPAAAP 12 54.9 187.5 2.3 Aliphatic 223 889ILVAAAPIAALP 12 57.3 195.8 2.2 Aliphatic 224 891 ILAVAAIPAALP 12 54.9195.8 2.2 Aliphatic 225 893 VIAIPAILAAAP 12 54.9 195.8 2.3 Aliphatic 226895 AIIIVVPAIAAP 12 50.2 211.7 2.5 Aliphatic 227 896 AILIVVAPIAAP 1250.2 211.7 2.5 Aliphatic 228 897 AVIVPVAIIAAP 12 50.2 203.3 2.5Aliphatic 229 899 AVVIALPAVVAP 12 57.3 195.0 2.4 Aliphatic 230 900ALVAVIAPVVAP 12 57.3 195.0 2.4 Aliphatic

TABLE 2-6 [Newly Developed Hydrophobic CPPs-240 aMTDs That All Critical Factors AreConsidered and Satisfied (Sequence ID No. 231-240)] Sequence Rigidity/Sturctural ID Flexibility Feature Hydropathy Residue Number aMTDSequences Length (II) (Al) (GRAVY) Structure 231 901 ALVAVLPAVAVP 1257.3 195.0 2.4 Aliphatic 232 902 ALVAPLLAVAVP 12 41.3 203.3 2.3Aliphatic 233 904 AVLAVVAPVVAP 12 57.3 186.7 2.4 Aliphatic 234 905AVIAVAPLVVAP 12 41.3 195.0 2.4 Aliphatic 235 906 AVIALAPVVVAP 12 57.3195.0 2.4 Aliphatic 236 907 VAIALAPVVVAP 12 57.3 195.0 2.4 Aliphatic 237908 VALALAPVVVAP 12 57.3 195.0 2.3 Aliphatic 238 910 VAALLPAVVVAP 1257.3 195.0 2.3 Aliphatic 239 911 VALALPAVVVAP 12 57.3 195.0 2.3Aliphatic 240 912 VALLAPAVVVAP 12 57.3 195.0 2.3 Aliphatic 52.6 ± 5.1201.7 ± 7.8 2.3 ± 0.1

These examined critical factors are within the range that we have setfor our critical factors; therefore, we were able to confirm that theaMTDs that satisfy these critical factors have much highercell-permeability (TABLE 3) and intracellular delivery potentialcompared to reference hydrophobic CPPs reported during the past twodecades.

TABLE 3 [Summarized Critical Factors of aMTD After In-Depth Analysis ofExperimental Results] Summarized Critical Factors of aMTD Analysis ofExperimental Results Critical Factor Range Bending Potential Prolinepresences in the middle (5′, 6′, 7′ or 8′) (Proline Position: PP) and atthe end (12′) of peptides Rigidity/Flexibility 41.3-57.3 (InstabilityIndex: II) Structural Feature 187.5-220.0 (Aliphatic Index: Al)Hydropathy 2.2-2.6 (Grand Average of Hydropathy GRAVY) Length 12 (Numberof Amino Acid) Amino acid Composition A, V, I, L, P2. Development of SOCS3 Recombinant Proteins Fused to aMTD andSolubilization Domain 2-1. Design of Novel Hydrophobic CPPs-aMTDs forDevelopment of Recombinant SOCS3 Proteins

Based on these six critical factors proven by experimental data, newlydesigned advanced macromolecule transduction domains (aMTDs) have beendeveloped, and optimized for their practical therapeutic usage tofacilitate protein translocation across the membrane. For this presentinvention, cell-permeable SOCS3 recombinant proteins have been developedby adopting aMTD165 (TABLE 4) that satisfied all 6 critical factors(TABLE 5).

TABLE 4  [Amino Acid and Nucleotide Sequence ofNewly Developed Advanced MTD 165 Which Follow All Critical Factors] IDAmino Acid Sequence Nucleotide Sequence 165 ALAVPVALAIVPGCG CTG GCG GTG CCG GTG GCG CTG GCG ATT GTG CCG

TABLE 5 [Critical Factors of aMTD165] Bending Potential Prolin Rigidity/Sturctural Theoretical M.W. Position Flexibility Feature Hydropathy IDLength pl (Da) 5′ 6′ 12′ (II) (Al) (GRAVY) 165 12 5.57 1133.4 — 1 1 50.2195.8 2.2

2-2. Selection of Solubilization Domain (SD) for 50053 RecombinantProteins

In the previous study, recombinant cargo (SOCS3) proteins fused tohydrophobic CPP could be expressed in bacteria system and purified withsingle-step affinity chromatography; however, protein dissolved inphysiological buffers (e.q. PBS, DMEM or RPMI1640 etc.) was highlyinsoluble and had extremely low. Therefore, an additional non-functionalprotein domain (solubilization domain: SD; TABLE 6) has been fused tothe recombinant proteins at their C terminus to improve lowsolubility/yield and to enhance relative cell-/tissue-permeability.

TABLE 6 [Information of Solublization Domains] Protein Instability SDGenbank ID Origin (kDa) pl Index (II) GRAVY A CP000113.1 Bacteria 23 4.648.1 −0.1 B BC086945.1 Pansy 11 4.9 43.2 −0.9 C CP012127.1 Human 12 5.830.7 −0.1 D CP012127.1 Bacteria 23 5.9 26.3 −0.1 E CP011550.1 Human 115.3 44.4 −0.9 F NG_034970 Human 34 7.1 56.1 −0.2

According to the specific aim, solubilization domain A (SDA) and B (SDB)were first selected. We hypothesize that fusion of SOCS3 with SDs andnovel hydrophobic CPP, aMTD, would greatly increase solubility/yield andcell-/tissue-permeability of recombinant cargo proteins—SOCS3—for theclinical application. SDA is a soluble tag, a tandem repeat of 2N-terminal domain (NTD) sequences of CP_(—)000113.1, which is a verystable soluble protein present in a spore surface coat of Myxococcusxanthus. SDB, a heme-binding part of cytochrome, provides a visual aidfor estimating expression level and solubility. Bacteria expressing SDBcontaining fusion proteins appears red when the fused proteins aresoluble.

2-3. Preparation of SOCS3 Recombinant Proteins

Histidine-tagged human SOCS3 proteins were designed (FIG. 1) andconstructed by amplifying the SOCS3 cDNA (225 amino acids) from nt 4 to678 using primers [TABLE 7] for SOCS3 cargo fused to aMTD. The PCRproducts were subcloned with NdeI (5′) and BamHI (3′) into pET-28a(+).Coding sequences for SDA or SDB were fused to the C terminus ofhis-tagged aMTD-fused SOCS3 and cloned at between the BamHI (5′) andSalI (3′) sites in pET-28a(+) (FIG. 2).

PCR primers for SOCS3 and SDA and/or SDB fused to SOCS3 are summarizedin TABLES 7, 8 and 9, respectively. The cDNA and amino acid sequences ofhistidine tag are provided in SEQ ID NO: 481 and 482, and cDNA and aminoacid sequences of aMTDs are indicated in SEQ ID NO: 483 and 484,respectively. The cDNA and amino acid sequences are displayed in SEQ IDNO: 485 and 486 (S0053); SEQ ID NO: 487 and 488 (SDA); and SEQ ID NO:489 and 490 (SDB), respectively.

[cDNA Sequence of Histidine Tag] SEQ ID NO: 481ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGC[Amino Acid Sequence of Histidine Tag] SEQ ID NO: 482Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser[cDNA Sequences of aMTDs] SEQ ID NO: 483 Please see TABLE 4[Amino Acid Sequences of aMTDs] SEQ ID NO: 484 Please see TABLE 4[cDNA Sequence of human SOC53] SEQ ID NO: 485ATGGTCACCC ACAGCAAGTT TCCCGCCGCC GGGATGAGCC GCCCCCTGGA CACCAGCCTGCGCCTCAAGA CCTTCAGCTC CAAGAGCGAG TACCAGCTGG TGGTGAACGC AGTGCGCAAGCTGCAGGAGA GCGGCTTCTA CTGGAGCGCA GTGACCGGCG GCGAGGCGAA CCTGCTGCTCAGTGCCGAGC CCGCCGGCAC CTTTCTGATC CGCGACAGCT CGGACCAGCG CCACTTCTTCACGCTCAGCG TCAAGACCCA GTCTGGGACC AAGAACCTGC GCATCCAGTG TGAGGGGGGCAGCTTCTCTC TGCAGAGCGA TCCCCGGAGC ACGCAGCCCG TGCCCCGCTT CGACTGCGTGCTCAAGCTGG TGCACCACTA CATGCCGCCC CCTGGAGCCC CCTCCTTCCC CTCGCCACCTACTGAACCCT CCTCCGAGGT GCCCGAGCAG CCGTCTGCCC AGCCACTCCC TGGGAGTCCCCCCAGAAGAG CCTATTACAT CTACTCCGGG GGCGAGAAGA TCCCCCTGGT GTTGAGCCGGCCCCTCTCCT CCAACGTGGC CACTCTTCAG CATCTCTGTC GGAAGACCGT CAACGGCCACCTGGACTCCT ATGAGAAAGT CACCCAGCTG CCGGGGCCCA TTCGGGAGTT CCTGGACCAGTACGATGCCC CGCTT [Amino Acid Sequence of human S0053] SEQ ID NO: 486Met Val Thr His Ser Lys Phe Pro Ala Ala Gly Met Ser Arg Pro Leu Asp Thr Ser Leu Arg Leu LysThr Phe Ser Ser Lys Ser Glu Tyr Gln Leu Val Val Asn Ala Val Arg Lys Leu Gln Glu Ser Gly Phe TyrTrp Ser Ala Val Thr Gly Gly Glu Ala Asn Leu Leu Leu Ser Ala Glu Pro Ala Gly Thr Phe Leu Ile ArgAsp Ser Ser Asp Gln Arg His Phe Phe Thr Leu Ser Val Lys Thr Gln Ser Gly Thr Lys Asn Leu Arg IleGln Cys Gly Gly Gly Ser Phe Ser Leu Gln Ser Asp Pro Arg Ser Thr Gln Pro Val Pro Arg Phe AspCys Val Leu Lys Leu Val His His Tyr Met Pro Pro Pro Gly Ala Pro Ser Phe Pro Ser Pro Pro Thr GluPro Ser Ser Glu Val Pro Glu Gln Pro Ser Ala Gln Pro Leu Pro Gly Ser Pro Pro Arg Arg Ala Tyr TyrIle Tyr Ser Gly Gly Glu Lys Ile Pro Leu Val Leu Ser Arg Pro Leu Ser Ser Asn Val Ala Thr Leu GlnHis Leu Cys Arg Lys Thr Val Asn Gly His Leu Asp Ser Tyr Glu Lys Val Thr Gln Leu Pro Gly Pro IleArg Glu Phe Leu Asp Gln Tyr Asp Ala Pro Leu [cDNA Sequences of SDA]SEQ ID NO: 487ATGGCAAATATT ACCGTTTTCTAT AACGAAGACTTC CAGGGTAAGCAG GTCGATCTGCCGCCTGGCAACTAT ACCCGCGCCCAG TTGGCGGCGCTG GGCATCGAGAAT AATACCATCAGCTCGGTGAAGGTG CCGCCTGGCGTG AAGGCTATCCTG TACCAGAACGAT GGTTTCGCCGGCGACCAGATCGAA GTGGTGGCCAAT GCCGAGGAGTTG GGCCCGCTGAAT AATAACGTCTCCAGCATCCGCGTC ATCTCCGTGCCC GTGCAGCCGCGC ATGGCAAATATT ACCGTTTTCTATAACGAAGACTTC CAGGGTAAGCAG GTCGATCTGCCG CCTGGCAACTAT ACCCGCGCCCAGTTGGCGGCGCTG GGCATCGAGAAT AATACCATCAGC TCGGTGAAGGTG CCGCCTGGCGTGAAGGCTATCCTC TACCAGAACGAT GGTTTCGCCGGC GACCAGATCGAA GTGGTGGCCAATGCCGAGGAGCTG GGTCCGCTGAAT AATAACGTCTCC AGCATCCGCGTC ATCTCCGTGCCGGTGCAGCCGAGG  [Amino Acid Sequences of SDA] SEQ ID NO: 488Met Ala Asn ile Thr Val Phe Tyr Asn Glu Asp Phe Gln Gly Lys Gln Val Asp Leu Pro Pro Gly AsnTyr Thr Arg Ala Gln Leu Ala Ala Leu Gly Ile Glu Asn Asn Thr Ile Ser Ser Val Lys Val Pro Pro GlyVal Lys Ala Ile Leu Tyr Gln Asn Asp Gly Phe Ala Gly Asp Gln Ile Glu Val Val Ala Asn Ala Glu GluLeu Gly Pro Leu Asn Asn Asn Val Ser Ser Ile Arg Val Ile Ser Val Pro Val Gln Pro Arg Met Ala AsnIle Thr Val Phe Tyr Asn Glu Asp Phe Gln Gly Lys Gln Val Asp Leu Pro Pro Gly Asn Tyr Thr ArgAla Gln Leu Ala Ala Leu Gly ile Glu Asn Asn Thr Ile Ser Ser Val Lys Val Pro Pro Gly Val Lys Ala Ile Leu Tyr Gln Asn Asp Gly Phe Ala Gly Asp Gln Ile Glu Val Val Ala Asn Ala Glu Glu Leu Gly Pro Leu Asn Asn Asn Val Ser Ser Ile Arg Val Ile Ser Val Pro Val Gln Pro Arg[cDNA Sequences of SDB] SEQ ID NO: 489ATGGCA GAACAAAGCG ACAAGGATGT  GAAGTACTAC ACTCTGGAGG AGATTCAGAAGCACAAAGAC AGCAAGAGCA CCTGGGTGAT CCTACATCAT AAGGTGTACG ATCTGACCAAGTTTCTCGAA GAGCATCCTG GTGGGGAAGA AGTCCTGGGC GAGCAAGCTG GGGGTGATGCTACTGAGAAC TTTGAGGACG TCGGGCACTC TACGGATGCA CGAGAACTGT CCAAAACATACATCATCGGG GAGCTCCATC CAGATGACAG ATCAAAGATA GCCAAGCCTT CGGAAACCCT T[Amino Acid Sequences of SDB] SEQ ID NO: 490Met Ala Glu Gln Ser Asp Lys Asp Val Lys Tyr Tyr Thr Leu Glu Glu Ile Gln Lys His Lys Asp Ser LysSer Thr Trp Val Ile Leu His His Lys Val Tyr Asp Leu Thr Lys Phe Leu Glu Glu His Pro Gly Gly GluGlu Val Leu Gly Glu Gln Ala Gly Gly Asp Ala Thr Glu Asn Phe Glu Asp Val Gly His Ser Thr Asp AlaArg Glu Leu Ser Lys Thr Tyr Ile Ile Gly Glu Leu His Pro Asp Asp Arg Ser Lys Ile Ala Lys Pro Ser Glu Thr Leu

The SOCS3 recombinant proteins were expressed in E. coli BL21-CodonPlus(DE3) cells, grown to an OD₆₀₀ of 0.6 and induced for 3 hrs with 0.6 mMisopropyl-β-D-thiogalactopyranoside (IPTG). The proteins were purifiedby Ni2⁺ affinity chromatography and dissolved in a physiological buffersuch as DMEM medium.

TABLE 7  [PCR Primers for His-Tagged SOCS3 Proteins] aMTD RecombinantCargo ID Protein 5′ Primers 3′ Primers SOCS3 — HS3 5′-GGAATTCCAT5′-CCCSGATCCT ATGGTCACCCACA TAAAGCGGGGCAT GCARGTTTCCCGC CGTACTGGTCCAGCGCC-3′ GAA-3′ 165 HM₁₆₅S3 5′-GGAATTCCAT 165 HM₁₆₅S3A ATGGCGCTGGCGG5′-CCGGATCCAA 165 HM₁₆₅S3B TGCCGGTGGCGCT GCGGGGCATCGTA GGCGATTGTGCCGCTGGTCCAGGAA-3′ GTCACCCACAGCA AGTTTC-3′ — HS3B 5′-GGAATTCCATATGGTCACCCACA GCAAGTTTCCCGC CGCC-3′

TABLE 8  [PCR Primers for aMTD/SDA-Fused SOCS3 Proteins] RecombinantCargo SD Protein 5′ Primers 3′ Primers SOCS3 SDA HM₁₆₅S3A5′-CCCGGATCCATG 5′-CGCGTCGACTTA GCAAATATTACCGTT CCTCGGCTGCACCGGTTCTATAACGAA-3′ CACGGCGATGAC-3′

TABLE 9  [PCR Primers for aMTD/SDB-Fused SOCS3 Proteins] RecombinantCargo SD Protein 5′ Primers 3′ Primers SOCS3 SDB HM₁₆₅S3B 5′-CCCGGATCCGC5′-CGCGTCGACTTA HS3B AGAACAAAGCGACA AAGGGTTTCCGAAGG AGGATGTGAAG-3′CTTGGCTATCTT-3′

2-4. Determination of Solubility and Yield of Each SOCS3 RecombinantProtein

The histidine-tagged SOCS3 proteins were expressed, purified, andprepared in soluble form (FIG. 3). The yield of each soluble SOCS3recombinant proteins was determined by measuring absorbance (A450).

SOCS3 recombinant proteins containing aMTD165 and solubilization domain(HM₁₆₅S3A and HM₁₆₅S3B) had little tendency to precipitate whereasrecombinant SOCS3 proteins lacking a solubilization domain (HS3 andHM₁₆₅S3) were largely insoluble. Solubility of aMTD/SD-fused SOCS3proteins was scored on a 5 point scale compared with that of SOCS3proteins lacking the solubilization domain (FIG. 4).

Yields per L of E. coli for each recombinant protein (mg/L) ranged from1 to 47 mg/L (FIG. 4). Yields of SOCS3 proteins containing an aMTD andSDB (HM₁₆₅S3B) were 50% higher than his-tagged SOCS3 protein (HS3).

3. aMTD/SD-Fused SOCS3 Recombinant Proteins Significantly Increase Cell-and Tissue-Permeability3-1. aMTD/SD-Fused 50053 Recombinant Proteins are Cell-Permeable

To examine protein uptake, SOCS3 recombinant proteins were conjugated to5/6-fluorescein isothiocyanate (FITC). RAW 264.7 (FIG. 5) or NIH3T3cells (FIG. 6) were treated with 10

M FITC-labeled SOCS3 recombinant proteins. The cells were washed threetimes with ice-cold PBS and treated with proteinase K to removesurface-bound proteins, and internalized proteins were measured by flowcytometry (FIG. 5) and visualized by confocal laser scanning microscopy(FIG. 6). SOCS3 proteins containing aMTD165 (HM165S3, HM165S3A andHM165S3B) efficiently entered the cells (FIGS. 5 and 6) and werelocalized to various extents in cytoplasm (FIG. 6). In contrast, SOCS3protein (HS3) containing lacking aMTD did not appear to enter cells.While all SOCS3 proteins containing aMTD165 transduced into the cells,HM165S3B displayed more uniform cellular distribution, and proteinuptake of HM165S3B was also very efficient.

3-2. aMTD/SD-Fused SOCS3 Recombinant Proteins Enhance the SystemicDelivery to a Variety of Tissues

To further investigate in vivo delivery of SOCS3 recombinant proteins,FITC-labeled SOCS3 proteins were monitored following intraperitoneal(IP) injections in mice. Tissue distributions offluorescence-labeled-SOCS3 proteins in different organs was analyzed byfluorescence microscopy (FIG. 7). SOCS3 recombinant proteins fused toaMTD165 (HM₁₆₅S3, HM₁₆₅S3A and HM₁₆₅S3B) were distributed to a varietyof tissues (liver, kidney, spleen, lung, heart and, to a lesser extent,brain). Predictably, liver showed highest levels of fluorescentcell-permeable SOCS3 since intraperitoneal administration favors thedelivery of proteins to this organ via the portal circulation. SOCS3containing aMTD165 was detectable to a lesser degree in lung, spleen andheart. aMTD/SDB-fused SOCS3 recombinant protein (HM₁₆₅S3B) showed thehighest systemic delivery of SOCS3 protein to the tissues comparable tothe SOCS3 containing only aMTD (HM₁₆₅S3) or aMTD/SDA (HM165S3A)proteins. These data suggest that SOCS3 protein containing both ofaMTD165 and SDB leads to higher cell-/tissue-permeability due to theincrease in solubility and stability of the protein, and it displayed adramatic synergic effect on cell-/tissue-permeability.

3-3. aMTD-Mediated Intracellular Delivery is Bidirectional Mode

SOCS3 recombinant proteins lacking SD (HS3 and HM165S3) were lesssoluble, produced lower yields, and showed tendency to precipitate whenthey were expressed and purified in E. coli. Therefore, we additionallydesigned (FIG. 8) and constructed SOCS3 recombinant protein containingonly SDB (without aMTD165: HS3B) as a negative control. As expected, itssolubility and yield increased compared to that of SOCS3 proteinslacking SDB (HS3; FIG. 9). Therefore, HS3B proteins were used as acontrol protein.

We next investigated how of aMTD165-mediated intracellular delivery wasoccurred. The aMTD-mediated intracellular delivery of SOCS3 protein didnot require protease-sensitive protein domains displayed on the cellsurface (FIG. 10B), microtubule function (FIG. 10C), or ATP utilization(FIG. 10D), since aMTD165-dependent uptake [compare to HS3 (black) andHS3B (blue)] was essentially unaffected by treating cells withproteinase K, taxol, or the ATP depleting agent, antimycin. Conversely,aMTD165-fused SOCS3 proteins uptake was blocked by treatment with EDTAand low temperature (FIGS. 10A and E), indicating the importance ofmembrane integrity and fluidity for aMTD-mediated protein transduction.

Moreover, we also tested whether cells treated with aMTD165-fused SOCS3protein could transfer the protein to neighboring cells. For this, cellstransduced with FITC-HM165S3B (green) were mixed with CD14-labeled cells(red), and cell-to-cell protein transfer was assessed by flow cytometry,scoring for CD14/FITC double-positive cells. Efficient cell-to-celltransfer of HM165S3B, but not HS3 or HS3B (FIG. 11), suggests that SOCS3recombinant proteins containing aMTD165 are capable of bidirectionalpassage across the plasma membrane.

4. aMTD/SD-Fused SOCS3 Protein Efficiently Inhibits Cellular Processes4-1. aMTD/SD-Fused SOCS3 Protein Inhibits the Activation of STATsInduced by INF-γ

The ultimate test of cell-penetrating efficiency is a determination ofintracellular activity of SOCS3 proteins transported by aMTD. Sinceendogenous SOCS3 are known to block phosphorylation of STAT1 and STAT3by IFN-γ-mediated Janus kinases (JAK) 1 and 2 activation, wedemonstrated whether cell-permeable SOCS3 inhibits the phosphorylationof STATs. All SOCS3 recombinant proteins containing aMTD (HM₁₆₅S3,HM₁₆₅S3A and HM₁₆₅S3B), suppressed IFN-γ-induced phosphorylation ofSTAT1 and STAT3 (FIG. 12). In contrast, STAT phosphorylation was readilydetected in cells exposed to HS3, which lacks the aMTD motif requiredfor membrane penetration (FIG. 12), indicating that HS3, which lacks anMTD sequence and did not enter the cells, has no biological activity.

4-2. aMTD/SD-Fused SOCS3 Recombinant Protein Inhibits the Secretion ofInflammatory Cytokines TNF-α and IL-6

We next investigated the effect of cell-permeable SOCS3 proteins oncytokines secretion. Treatment of C3H/HeJ primary peritoneal macrophageswith SOCS3 proteins containing aMTD165 suppressed TNF-α and IL-6secretion induced by the combination of IFN-γ and LPS by 50-90% duringsubsequent 9 hrs of incubation (FIG. 13). In particular,aMTD165/SDB-fused SOCS3 recombinant protein showed the greatestinhibitory effect on cytokine secretion. In contrast, cytokine secretionin macrophages treated with non-permeable SOCS3 protein (HS3) wasunchanged, indicating that recombinant SOCS3 lacking the aMTD doesn'taffect intracellular signaling. Therefore, we conclude that differencesin the biological activities of HM₁₆₅S3B as compared to HS3B are due tothe differences in protein uptake mediated by the aMTD sequence. Inlight of solubility/yield, cell-/tissue-permeability, and biologicaleffect, SOCS3 recombinant protein containing aMTD and SDB (HM₁₆₅S3B) isa prototype of a new generation of improved cell-permeable SOCS3(iCP-SOCS3), and will be selected for further evaluation as a potentialanti-tumor agent.

5. iCP-SOCS3 Suppresses Pro-Tumorigenic Functions in Lung Cancer Cells5-1. iCP-50053 Enhances the Cellular Uptake into Lung Cancer Cells andthe Systemic Delivery to the Lung Tissue

Although lung cancer is one of the most common cancers with a highmortality rate, there are few drugs for treating this lethal disorder.Since constitutive activation of STAT3 is found in various cancers andSOCS3 is closely related to the development of lung cancer, we firstchose lung cancer as a primary indication of the iCP-SOCS3 as ananti-cancer agent.

To determine the cell-permeability of iCP-SOCS3 in the lung cancercells, cellular uptake of FITC-labeled SOCS3 recombinant proteins wasquantitatively evaluated by flow cytometry. FITC-HM₁₆₅S3B recombinantprotein (iCP-SOCS3) promoted the transduction into cultured A549 lungcancer cells (FIG. 14). In addition, iCP-SOCS3 proteins enhanced thesystemic delivery to lung tissue after intraperitoneal injection (FIG.15). Therefore, these data indicate that iCP-SOCS3 protein could beintracellularly delivered and distributed to the lung cells and tissue,contributing for beneficial biotherapeutic effects.

5-2. iCP-50053 Inhibits Viability of Lung Cancer Cells

Since the endogenous level of SOCS3 protein is reduced in lung cancerpatients, and SOCS3 negatively regulates cell growth and motility incultured lung cancer cells, we investigated whether iCP-SOCS3 inhibitscell viability through SOCS3 intracellular delivery in lung cancercells. As shown in FIG. 16, SOCS3 recombinant proteins containingaMTD165 significantly suppressed cancer cell proliferation. HM₁₆₅S3B(iCP-SOCS3) protein was the most cytotoxic to A549 lung cancercells—over 80% in 10 μM treatment (p<0.01)—especially compared tovehicle alone (i.e. exposure of cells to culture media withoutrecombinant proteins; FIG. 16, left). However, neither cell-permeableSOCS3 protein adversely affected the cell viability of non-cancer cells(NIH3T3) even after exposing these cells to equal concentrations (10 μM)of protein over 4 days (FIG. 16, right). These results suggest that theiCP-SOCS3 protein is not overly toxic to normal cells and selectivelykills cancer cells, and would have a great ability to inhibit cellsurvival-associated phenotypes in lung cancer without any severeaberrant effects as a protein-based biotherapeutics.

5-3. iCP-SOCS3 Protein Induces Apoptosis in Lung Cancer Cells

To further determine the effect of iCP-SOCS3 on the tumorigenicity oflung cancer cells, we subsequently investigated whether iCP-SOCS3regulates apoptosis in A549 cells. HM₁₆₅S3B protein (iCP-SOCS3) was aconsiderably efficient inducer of apoptosis in A549 cells, as assessedeither by a fluorescent terminal dUTP nick-end labeling (TUNEL) assay(FIG. 17) and Annexin V staining (FIG. 18). Consistently, no changes inTUNEL and Annexin V staining were observed in A549 cells treated withHS3B compared to untreated cells (Vehicle).

5-4. iCP-SOCS3 Inhibits Migration/Invasion of Lung Cancer Cells

We next examined the ability of iCP-SOCS3 to influence cell migration.A549 cells were treated with recombinant proteins for 2 hrs, themonolayers were wounded, and cell migration in the wound was monitoredafter 48 hrs (FIG. 19). HM₁₆₅S3B protein (iCP-SOCS3) suppressed therepopulation of wounded monolayer although SOCS3 protein lacking aMTD165(HS3B) had no effect on the cell migration. Consistent with this, A549cells treated with HM₁₆₅S3B recombinant protein (iCP-SOCS3) also showedsignificant inhibitory effect on their Transwell migration compared withuntreated cells (Vehicle) and non-permeable SOCS3 protein-treated cells(HS3B; FIG. 20). In addition, A549 cells treated with HM₁₆₅S3Brecombinant protein (iCP-SOCS3) caused remarkable decrease in invasioncompared with the control proteins (HS3B; FIG. 21). Taken together,these data indicate that iCP-SOCS3 contributes to inhibit tumorigenicactivities of lung cancer cells.

Example

The following examples are presented to aid practitioners of theinvention, to provide experimental support for the invention, and toprovide model protocols. In no way are these examples to be understoodto limit the invention.

Example 1 Development of Novel Advanced Macromolecule TransductionDomain (aMTD)

H-regions of signal sequences (HRSP)-derived CPPs (MTM, MTS and MTD) donot have a common sequence, a sequence motif, and/or a common structuralhomologous feature. In this invention, the aim is to develop improvedhydrophobic CPPs formatted in the common sequence and structural motifthat satisfy newly determined ‘critical factors’ to have a ‘commonfunction’, to facilitate protein translocation across the membrane withsimilar mechanism to the analyzed CPPs. 6 critical factors have beenselected to artificially develop novel hydrophobic CPP, namely advancedmacromolecule transduction domain (aMTD). These 6 critical factorsinclude the followings: amino acid length of the peptides (ranging from9 to 13 amino acids), bending potentials (dependent with the presenceand location of proline in the middle of sequence (at 5′, 6′, 7′ or 8′amino acid) and at the end of peptide (at 12′)), instability index (II)for rigidity/flexibility (II: 40-60), grand average of hydropathy(GRAVY) for hydropathy (GRAVY: 2.1-2.4), and aliphatic index (AI) forstructural features (AI: 180-220). Based on these standardized criticalfactors, new hydrophobic peptide sequences, namely advancedmacromolecule transduction domain peptides (aMTDs), in this inventionhave been developed and selected to be fused with the cargo protein,SOCS3, to develop improved cell-permeable SOCS3 recombinant protein(iCP-SOCS3).

Example 2 Construction of Expression Vectors for Recombinant SOCS3Proteins

Histidine-tagged human SOCS3 proteins were constructed by amplifying theSOCS3 cDNA (225 amino acids) for aMTD fused to SOCS3 cargo. The PCRreactions (100 ng genomic DNA, 10 pmol each primer, each 0.2 mM dNTPmixture, lx reaction buffer and 2.5 U Pfu(+) DNA polymerase (Doctorprotein, Korea)) were digested on the restriction enzyme site betweenNde I (5′) and Sal I (3′) involving 35 cycles of denaturing (95° C.),annealing (62° C.), and extending (72° C.) for 45 sec each. For the lastextension cycle, the PCR reactions remained for 10 min at 72° C. The PCRproducts were subcloned into 6×His expression vector, pET-28a(+)(Novagen). Coding sequence for SDA or SDB fused to C terminus ofhis-tagged aMTD-SOCS3 was cloned at BamHI (5′) and SalI (3′) inpET-28a(+) from PCR-amplified DNA segments and confirmed by DNA sequenceanalysis of the resulting plasmids.

Example 3 Inducible Expression, Purification, and Preparation ofRecombinant Proteins

The recombinant proteins were purified from E. coli BL21-CodonPlus (DE3)cells grown to an A600 of 0.6 and induced for 3 hrs with 0.6 mM IPTG.Denatured recombinant proteins were purified by Ni2+ affinitychromatography as directed by the supplier (Qiagen, Hilden, Germany).After purification, they were dialyzed against a refolding buffer (0.55M guanidine HCl, 0.44 M L-arginine, 50 mM Tris-HCl, 150 mM NaCl, 1 mMEDTA, 100 mM NDSB, 2 mM reduced glutathione, and 0.2 mM oxidizedglutathione) and changed to a physiological buffer such as DMEM medium.

Example 4 Determination of Quantitative Cell-Permeability of RecombinantProteins

For quantitative cell-permeability, recombinant SOCS3 proteins wereconjugated to 5/6-fluorescein isothiocyanate (FITC) according to themanufacturer's instructions (Sigma-Aldrich, St. Louis, Mo.). RAW 264.7cells were treated with 10 μM FITC-labeled recombinant proteins for 1 hrat 37° C., washed three times with cold PBS, and treated with proteinaseK (10 μg/mL) for 20 min at 37° C. to remove cell-surface bound proteins.Cell-permeability of these recombinant proteins was analyzed by flowcytometry (Guava, Millipore, Darmstadt, Germany) using the FlowJocytometric analysis software.

Example 5 Determination of Intracellular Localization of SOCS3Recombinant Proteins

For visual cell permeability, NIH3T3 cells were cultured on coverslipsin 24-well plates and with 10 μM FITC-conjugated recombinant proteinsfor 1 hr at 37° C. These cells on coverslips were washed with PBS, fixedwith 4% formaldehyde for 10 min, and washed three times with PBS at roomtemperature. Coverslips were mounted with VECTASHIELD Mounting Medium(Vector laboratories, Burlingame, Calif.) with DAPI(4′,6-diamidino-2-phenylindole) for nuclear staining. Intracellularlocalization of fluorescent signal was determined by confocal laserscanning microscopy (LM700, Zeiss, Germany).

Example 6 Determination of Tissue Distribution of Recombinant SOCS3Proteins

ICR mice (6-week-old, female) were injected intraperitoneally (600μg/head) with either FITC only or FITC-conjugated SOCS3 recombinantproteins. After 2 hrs, the liver, kidney, spleen, lung, heart, and brainwere isolated, washed with an O.C.T. compound (Sakura), and frozen ondry ice. Cryosections (20 μm) were analyzed by fluorescence microscopy(Carl Zeiss, Gottingen, Germany).

Example 7 Mechanism of aMTD-Mediated Intracellular Delivery

RAW264.7 cells were pretreated with different agents to assess theeffect of various conditions on protein uptake: (i) 5 μg/ml proteinase Kfor 10 min, (ii) 20 μM Taxol for 30 min, (iii) 10 μM antimycin in thepresence or absence of 1 mM ATP for 2 hrs, (iv) incubation on ice (ormaintained at 37° C.) for 60 min, and (v) 100 mM EDTA for 3 hrs. Theseagents were used at concentrations known to be active in otherapplications. The cells were then incubated with 10 μM FITC-labeledproteins for 1 hr at 37° C., washed three times with ice-coldphosphate-buffered saline, treated with proteinase K (10 μg/ml for 5 minat 37° C.) to remove cell-surface bound proteins, and analyzed by flowcytometry. To assess cell-to-cell protein transfer, RAW264.7 cellscontaining FITC-conjugated protein were prepared in the same way andmixed with untreated cells labeled with PreCP-Cy5.5-CD14 antibody for 2hrs. Cell-to-cell protein transfer, resulting in FITC-Cy5.5double-positive cells, was monitored by flow cytometry.

Example 8 STAT Phosphorylation: Western Blot Analysis

PANC-1 cells (Korean Cell Line Bank, Seoul, Korea) were cultured inmodified Eagle's medium (DMEM; Welgene, Daege, Korea) supplemented with10% (v/v) FBS, penicillin (100 units/ml), and streptomycin (10

g/ml, Gibco BRL) and pretreated with 10

M of SOCS3 recombinant proteins for 2 hrs followed by exposing the cellsto agonists (100 ng/ml IFN-

) for 15 min. Cells were lysed with RIPA lysis buffer (50 mM Tris pH8.0, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate,10 mM NaF, and 2 mM Na3VO4) containing a protease inhibitor cocktail andthen centrifuged at 13,000×g for 15 min at 4° C. Equal amounts oflysates were resolved by SDS-PAGE, transferred onto PVDF membranes, andprobed with phospho (pY701)-specific STAT1 (Cell Signaling, Danvers,Mass.).

Example 9 Cytokine Measurement: Cytometric Bead Array (CBA) Assay

Peritoneal macrophages were obtained from C3H/HeJ mice. Peritonealmacrophages were incubated with 10 μM recombinant proteins (1:HS3,2:HM165S3, 3:HM165S3A and 4:HM165S3B, respectively) for 1 hr at 37° C.and then stimulated them with LPS (500 ng/ml) and/or IFN-

(100 U/ml) without removing iCP-SOCS3 proteins for 3, 6, or 9 hrs. Theculture media were collected, and the extracellular levels of cytokinewere measured by a cytometric bead array (BD Biosciences, Pharmingen)according to the manufacturer's instructions.

Example 10 Cell Proliferation: CellTiter-Glo Cell Viability Assay

Cells originated from lung cancer and mouse fibroblast (NIH3T3) werepurchased (ATCC, Manassas, Va.) and maintained as recommended by thesupplier. These cells (3×103/well) were seeded in 96 well plates. Thenext day, cells were treated with DMEM (vehicle) or recombinant SOCS3proteins for 96 hrs in the presence of serum (2%). Proteins werereplaced daily. Cell growth and survival were evaluated with theCellTiter-Glo Cell Viability Assay (Promega, Madison, Wis.).Measurements using a Luminometer (Turner Designs, Sunnyvale, Calif.)were conducted following the manufacturer's protocol.

Example 11 Apoptosis: TUNEL Assay

Apoptotic cells were analyzed using terminal dUTP nick-end labeling(TUNEL) assay with In Situ Cell Death Detection kit TMR red (Roche, 4056Basel, Switzerland). Cells were treated with either 10 μM SOCS3recombinant protein or buffer alone for 16 hrs with 2% fetal bovineserum. Treated cells were washed with cold PBS two times, fixed in 4%paraformaldehyde (PFA, Junsei, Tokyo, Japan) for 1 hr at roomtemperature, and incubated in 0.1% Triton X-100 for 2 min on the ice.Cells were washed with cold PBS twice, and treated TUNEL reactionmixture for 1 hr at 37° C. in dark, washed cold PBS three times andobserved by fluorescence microscopy (Nikon, Tokyo, Japan).

Example 12 Apoptosis: Annexin V/7-AAD Staining

Annexin V/7-Aminoactinomycin D (7-AAD) staining was performed using flowcytometry according to the manufacturer's guidelines. Briefly, 1×106cells were washed three times with ice-cold PBS. The cells were thenresuspended in 100 μl of binding buffer and incubated with 1 μl of 7-AADand 1 μl of annexin V-PE for 30 min in the dark at 37° C. Flowcytometric analysis was immediately performed using a guava easyCyte™ 8Instrument (Merck Millipore).

Example 13 Cell Migration: Wound-Healing Assay

Cells were seeded into 12-well plates, grown to 90% confluence, andincubated with 10 μM HS3, HM16553, HM165S3A or HM165S3B in serum-freemedium for 2 hrs prior to changing the growth medium. The cells werewashed twice with PBS, and the monolayer at the center of the well was“wounded” by scraping with a pipette tip. Cells were cultured for anadditional 48 hrs and cell migration was observed by phase contrastmicroscopy. The migration is quantified by counting the number of cellsthat migrated from the wound edge into the clear area.

Example 14 Transwell Migration Assay

The lower surface of Transwell inserts (Costar) was coated with gelatin(10

g/ml), and the membranes were allowed to dry for 1 hr at roomtemperature. The Transwell inserts were assembled into a 24-well plate,and the lower chamber was filled with growth media containing 10% FBSand FGF2 (10

g/ml). Cells (5×105) were added to each upper chamber, and the plate wasincubated at 37° C. in a 5% CO2 incubator for 24 hrs. Migrated cellswere stained with 0.6% hematoxylin and 0.5% eosin and counted.

Example 15 Invasion Assay

The lower surface of Transwell inserts (Costar) was coated with gelatin(10

g/ml), the upper surface of Transwell inserts was coated with matrigel(40

g per well; BD Biosciences), and the membranes were allowed to dry for 1hr at room temperature. The Transwell inserts were assembled into a24-well plate, and the lower chamber was filled with growth mediacontaining 10% FBS and FGF2 (10

g/ml). Cells (5×105) were added to each upper chamber, and the plate wasincubated at 37° C. in a 5% CO2 incubator for 24 hrs. Migrated cellswere stained with 0.6% hematoxylin and 0.5% eosin and counted.

Example 16 Statistical Analysis

All data are presented as mean±s.d. Differences between groups weretested for statistical significance using Student's t-test and wereconsidered significant at p<0.05 or p<0.01.

It will be apparent to those skilled in the art that variousmodifications can be made to the above-described exemplary embodimentsof the present invention without departing from the spirit or scope ofthe invention. Thus, it is intended that the present invention coversall such modifications provided that they come within the scope of theappended claims and their equivalents.

What is claimed is:
 1. The list of amino acid sequences of SOCS3recombinant proteins fused to newly invented hydrophobiccell-penetrating peptides (CPPs)—advanced macromolecule transductiondomains (aMTDs) and solubilization domain (SD).
 2. The list of cDNAsequences of the SOCS3 recombinant proteins fused to newly inventedhydrophobic cell-penetrating peptides (CPPs), namely advancedmacromolecule transduction domains (aMTDs) and solubilization domain(SD) of claim
 1. 3. A list of 240 aMTD amino acid sequences according toclaim 1 that satisfy all six critical factors as shown in TABLE 3 4.Varied numbers and locations of solubilization domain (SD) according toclaim 1 that are fused to SOCS3 recombinant proteins for high solubilityand yield
 5. The result of therapeutic applicability in lung cancer withSOCS3 recombinant proteins fused to newly invented hydrophobiccell-penetrating peptides (CPPs), namely advanced macromoleculetransduction domains (aMTDs) and solubilization domain (SD)